Gamma-II-crystallin belongs to the family of beta-gamma-crystallins and is a structural protein of the eye lens with ubiquitary distribution in vertebrates (Jaenicke & Slingsby, 2001). Beta-gamma-crystallins form a highly homologous protein family characterized by two structurally identical domains and consisting largely of beta sheet structures (Wistow & Piatigorsky, 1988). The superimposed structural motif of the beta-gamma-crystallins is the so-called greek key topology. It consists of four antiparallel beta strands two of which—lying one over the other—form a domain of the crystallins (Blundell et al., 1981).
The natural function of the crystallins is based on the generation of a high refractive index in the lens of the eye which is achieved by an extremely high local protein concentration of up to 860 mg/ml (Kumaraswamy et al., 1996). Due to their spatial structure, crystallins are very stable and readily soluble proteins having a high protease resistance. Furthermore, the localization in the interior of the eye lens has the effect that gamma-crystallins are not subject to protein turnover. Therefore, beta-gamma-crystallins have one of the highest half-lives known for proteins (Jaenicke, 1996).
The best characterized member of this protein family is bovine gamma-crystallin. The spatial structure could be determined for the wild type of the protein at different resolutions as well as for a whole range of point mutants (Najmudin et al., 1993; Kumaraswamy et al., 1996; Norledge et al., 1996). This revealed that the protein is stabilized via a hydrophobic cleft between the two domains. This cleft is formed by intramolecular interactions of six hydrophobic residues consisting of three residues in the N-terminal domain and the three topologically identical residues in the C-terminal domain (Wistow et al., 1983). Die stability to chemical agents is largely independent of the short peptide linking the two domains (Mayr et al., 1994).
Bovine gamma-crystallin has a size of approx. 20 kDa and is characterized by an extraordinarily high stability. It is resistant to 8 M urea at a neutral pH. In a pH range of 1 to 9 it is present in its native state (Rudolph et al., 1990; Sharma et al., 1990), and even up to a temperature of 75° C. the protein is stable in 7 M urea (Jaenicke, 1994). The recombinant cytosolic expression of gamma-crystallins in E. coli is successful with very high yields (Mayr et al., 1994).
The protein-chemical properties—high stability, low molecular weight, high cytosolic expression rates—makes the protein class of gamma-crystallins attractive candidates for the generation of alternative binding molecules.
A phagemide library (GCUC1) has been established by Fiedler & Rudolph on the basis of the bovine gamma-II-crystallin as a scaffold protein wherein eight surface-exposed amino acids at positions 2, 4, 6, 15, 17, 19, 36 and 38 (without the starting methionine) were randomized on the DNA level. After several rounds of selection by means of phage display, variations with specific binding to estradiol and an affinity in the μM range could be detected. These results have shown that binding properties which did not exist before can be generated de novo on the bovine gamma-II-crystallin and that gamma-crystallins are generally suitable as scaffold proteins (scaffold) for the isolation of alternative binding molecules (see patent DE19932688 A1).
In subsequent works a new library was established on the basis of the human gamma-II-crystallin. The selection of the human gamma-II-crystallin as the scaffold and the accompanying construction of a new library hat important advantages: 1. Compared to the bovine protein the human gamma-II-crystallin has a significantly higher stability to denaturing influences, 2. the human origin of the protein should result in a very low immunogenicity of the respective variations in therapeutic applications and 3. a newly constructed library having a higher complexity should enable the isolation of binding molecules with higher affinity (Ling 2003). Similar to the GCUC1 library the same eight amino acid positions (without the starting methionine position 2, 4, 6, 15, 17, 19, 36, 38) were selected for randomization. The new library CR20 established according to described methods (patent DE19932688 A1) on the basis of the human gamma-II-crystallin has a theoretical size of 5×108 independent variations each of which is represented about 130 times in the library. After sequencing of more than 200 independent variations it was found that more than 80% of all variations had substitutions only in the eight randomized positions. Furthermore, the sequences of the variations in the substituted positions except the third codon position showed an almost identical distribution of all possible nucleotides. Thus, this library which has all 32 possible codons in the eight randomized positions is of high quality.
As a second scaffold protein for the generation of alternative binding molecules use is made of human ubiquitin. Ubiquitin is a small, monomeric and cytosolic protein which—highly conserved in its sequence—is present in all known eukaryotic cells from protozoa to vertebrates. In the organism, it plays a fundamental role in the regulation of the controlled degradation of cellular proteins.
The polypeptide chain of ubiquitin consists of 76 amino acids which are folded in an extraordinarily compact alpha/beta structure (Vijay-Kumar, 1987): Almost 87% of the polypeptide chain are involved in the formation of the secondary structural elements via hydrogen bonds. As the prominent is secondary structures three and a half alpha-helical turns as well as an antiparallel beta sheet consisting of five strands can be mentioned. The characteristic arrangement of these elements—an antiparallel beta sheet exposed to a surface of the protein onto the back side of which an alpha helix is packed which lies vertically on top of it—is generally considered as so-called ubiquitin-like folding motif. Another structural feature is a marked hydrophobic region in the interior of the protein between the alpha helix and the beta sheet.
Because of its small size, the artificial preparation of ubiquitin can be carried out both by chemical synthesis and by means of biotechnological methods. Due to the favorable folding properties, ubiquitin can be produced by genetic engineering using microorganisms such as e.g. E. coli in relatively large amounts either in the cytosol or in the periplasmic space.
Due to the simple and efficient bacterial preparation, ubiquitin can be used as a fusion partner for other foreign proteins to be prepared the production of which is problematic. By means of the fusion to ubiquitin an improved solubility and thereby an improved yield can be achieved (Butt et al., 1989).
On the basis of available data on the crystal structure (PDB data base entry 1UBI) using computerized analysis the positions of those amino acids in the ubiquitin protein scaffold could be localized the side chains of which are exposed to the surface i.e. to the solvent or a potential binding partner. The positions selected are localized in spatial proximity to each other at the beginning of the first aminoterminal beta sheet strand (pos. 2, 4, 6) as well as in the loop (pos. 62, 63) and at the beginning of the carboxyterminal beta sheet strand (pos. 64, 65, 66), respectively, forming with their amino acid side chains a contiguous region on the surface of ubiquitin. In this way, a surface-exposed hypervariable region was generated by random amino acid substitutions in the analyzed region on the still intact protein structure of ubiquitin (PCT/EP2004/005730, unpublished).
The generation of an artificial binding surface on a beta sheet protein represents a novel and interesting alternative to conventional antibodies. Evidence was obtained that a new, artificially generated binding site on the surface of gamma-crystallins or ubiquitin-like proteins results in functional binding molecules (DE 19932688 A1) (PCT/EP2004/005730, unpublished).
However, up to now there was no suggestion or indication as to the coupling of these polypeptide molecules to another component to form a conjugate rendering them useful for diagnostic, therapeutic and analytic applications without encountering a loss of function of one of the two or of both components.